The rate-limiting step of cholesterol biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, HMGR). This enzyme plays a critical role in cholesterol homeostasis, particularly in liver - the major site of regulatable cholesterol synthesis in the body. Recent studies demonstrating that intrinsic resistance to the serum and tissue cholesterol raising action of dietary cholesterol is proportional to the level of expression of hepatic HMGR make knowledge of the physiological mechanisms of molecular regulation of this enzyme very important. In contrast with tumor/transformed cell HMGR, feedback regulation of hepatic HMGR by dietary occurs primarily at the level of translation. Three specific aims for physiological regulation of hepatic HMGR are proposed in the Research Plan. They are: 1) translational regulation by dietary cholesterol, 2) transcriptional regulation by insulin, and 3) transcriptional and mRNA stabilization effects mediated by thyroid hormone. The possibilities that dietary may act to decrease translation of hepatic HMGR mRNA by inducing the synthesis of a protein that might bind to the stem-loop structure of the 5'untranslated region of the mRNA or by acting to increase levels of an oxylanosterol which would bind to a protein to slow translation will both be investigated. Gel shift, reporter construct assays, protein purification and a lipidomics investigation using HPLC and GC/MS will be conducted to examine these possibilities. Candidate insulin response elements will be investigated by introducing HMGR DNA regions ligated to luciferase directly into livers of diabetic or insulin-treated diabetic rats by in vivo electroporation or by hydrodynamic tail vein injection. The effects of relationship between hepatic HMGR expression and resistance to dietary cholesterol will be examined by either over expressing hepatic HMGR or by knocking it down with hydrodynamic tail vein injections of plasmids containing HMGR cDNA or siRNA. Regions of the HMGR gene that may mediate T3 stimulation of HMGR transcription will also be investigated by in vivo electroporation. The identity of possible T3-induced transcription factors will be sought using microarray analysis. RNA gel shift, protein purification, 3'UTR reporter construct studies, microarray analysis, and RT-PCR analysis will be used to identify protein(s) that likely mediate mRNA stability effects. These proposed investigations should provide significant insight into the molecular basis by the major physiological factors regulate expression of hepatic HMGR. Public Health Relevance: The results of the proposed research may lead to a greater appreciation for the beneficial role of cholesterol production by the liver in helping to achieve desirable cholesterol levels and lead to strategies to increase liver's capacity for cholesterol production to attain lower LDL cholesterol levels. A good understanding of the molecular basis by which dietary cholesterol and the body's major intrinsic regulators, the hormones - insulin and thyroid hormone, act to regulate cholesterol production will likely emerge from the experiments proposed in this study.